A Study Of Killing Effect Of CAR-T Cells Mediated By Anti-FGFR4 On Hepatoma Cells

BACKGROUNDIn recent years,as the incidence of cancer has increased year by year,there is an urgent need for effective treatments of cancer in the clinic.The specific immunotherapy has received increasing attention.Among them,Chemical antigen receptor T cells(CAR-T)therapy,which belongs to the specific T cell-based immunotherapy,has considered to be one of the most promising ways to treat cancer.The chimeric antigen receptor can specifically recognize and bind tumor-associated antigen targets without restriction of MHC and activate T lymphocytes.Thereby they effectively kill tumor cells.At present,CAR technology has been developed from the first to the fourth generation,TRUCK(T cells redirected for universal cytokine-mediated killing),which has achieved remarkable results in the treatment of hematological tumors.In the future it is likely to achieve effective treatment of solid tumors.Fibroblast growth factor receptor 4(FGFR4)is a major FGF receptor subtype in the liver and is highly expressed in liver cancer cells.Hepatocellular carcinoma(HCC)progression can be promoted by FGFR4,which regulats the secretion of alpha-fetoprotein(AFP),promoting proliferation and anti-apoptosis.Moreover,clinical studies have found that the expression of FGFR4 is also up-regulated in prostate cancer,breast cancer,pancreatic cancer and other tumors,and the high expression of FGFR4 is associated with tumor invasion and resistance to radiotherapy and chemotherapy.These results show that FGFR4,which is highly expressed on the surface of liver cancer cells,can be used as a potential target for tumor therapy through designing CAR-T against liver cancer.OBJECTIVEIn this study,a CAR lentiviral vector targeting FGFR4 was constructed and packaged into lentiviral particles.The mock CAR-T cells were prepared by transfecting human T lymphocyte leukemia cells(Jurkat cells).The killing effects on FGFR4-positive hepatoma cells HepG2 and SMMC-7721 were observed and measured.This is a preliminary study on the effect of mimic CAR-T cells killing FGFR4-positive hepatoma cells in vitro and in vivo.It will start an avenue for original CAR-T construction and in vivo application research,as well as provide a new immunotherapeutic strategy for liver cancer.METHODSThis study by fusing the light and heavy chain variable regions which had been previously screened out by the antibody library into anti-FGFR4 scFv.The anti-FGFR4 scFv was used as a single-chain variable region protein of extracellular antibody,CD28 as a transmembrane protein,a co-stimulatory molecule CD137 intracellular domain and a T cell receptor CD3?chain as an intracellular signaling domain to construct a chimeric antigen receptor anti-FGFR4-CAR.The anti-FGFR4-CAR fragment was cloned into the lentiviral expression vector pCDH-CMV-MCS-EF1-copEGFP-T2A-Puro to construct chimeric antigen receptor lentiviral expression vector pCDH-FGFR4-CAR.Double enzyme digestion and DNA sequencing show the sequence is correct.The lentiviral expression plasmid pCDH-FGFR4-CAR and two lentiviral packaging plasmids psPAX2 and pMD2.G were co-transfected into Lenti-X-293T cells to package lentivirus particles.The virus titer was determined by well dilution method.Finally,the Jurkat cells were infected with lentivirus particles,and the infection efficiency was detected by flow cytometry.The proliferation and killing effect of FGFR4-CAR-Jurkat cells were detected by the CCK-8 proliferation assay and lactate dehydrogenase release assay respectively.RESULTS1.The lentiviral expression vector pCDH-FGFR4-CAR was successfully constructed,through DNA sequencing analysis and identification of double enzyme digestion.2.The lentivirus particle was successfully packaged by the three-plasmid co-transfection method,and the virus titer was determined upto 4x10~8 TU/ml by the well dilution method.3.The simulated CAR-T cells/anti-FGFR4-Jurkat cells had been successfully established and the virus infection rate reached 92.44%.4.The FGFR4 antigen molecule can stimulate the proliferation of FGFR4-CAR-Jurkat cells.5.Along with the increase of the ratio of effective cells to target cells,the killing ability of FGFR4-CAR-Jurkat on liver cancer cells gradually increased.CONCLUSIONWe successfully constructed the third-generation FGFR4 chimeric antigen receptor lentiviral expression vector pCDH-FGFR4-CAR,and successfully packaged the lentivirus particles pCDH-FGFR4-CAR;The constructed FGFR4-CAR-Jurkat cells can be stably cultured and expanded in vitro.Compared to the control,the proliferation of FGFR4-CAR-Jurkat cells were significantly enhanced by stimulation with FGFR4 on the surface of the plates.In addition,compared to the normal hepatocytes,the FGFR4-CAR-Jurkat cells were able to specifically recognize FGFR4 molecules and kill the FGFR4-beared hepatoma cells HepG2 and SMMC-7721.NOTE:The anti-FGFR4 scFv sequence will not presented in the thesis due to patent...